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Plenary Presentation: History of orchid propagation

Joseph Arditti*
University of California, 4203, 5438 BS II, Mail Code: 2300, Irvine, CA 92697, USA.
Proceedings Asia Pacific Conference on Plant Tissue and Agribiotechnology (APaCPA) 17-21 June 2007

A good argument can be made that orchid multiplications procedures were always innovative when compared to the plant propagation biotechnology of their time (Arditti, 1984, 1990, 2007) . Seed Germination. The first method for orchid seed germination (for reviews see Arditti, 1984, 2007; Yam et al., 2002) was novel and very different from the methods used to germinate other seeds 155 years ago. David Moores (1807-1879) approach was new and an important advance in horticulture and biology. Exactly 50 years after Moore made his discovery Nol Bernard (1874-1911) made another major innovation when he formulated his procedure for symbiotic germination of orchid seeds in vitro (Arditti, 1984, 1990; Rasmussen, 1995; Yam et al., 2002). This may have been the first method for plant propagation in vitro. It was based on what were modern and advanced microbiological procedures at that time. The asymbiotic method for orchid seed germination developed by Lewis Knudsons (1884-1958; (for reviews see Arditti, 1984, 1990; Yam et al., 2002) was the first procedure for in vitro propagation of any plant in pure (i.e. axenic) culture. This method was a major methodological, biological and technological advance which presaged modern biotechnology. Knudson's first solution, known as the Knudson B medium (KB) was a modification of Pfeffers Solution, a formulation devised by the German plant physiologist Wilhelm Pfeffer (1845-1920). It was, and still is, a reasonably good medium for orchid seed germination, but Knudson improved it and published his solution C (Knudson C, KC) in 1946. This medium is used very widely for orchid seed germination (Arditti et al., 1982) and the micropropagation of some orchids. Micropropagation of orchids: The first attempt. Just before the end of the 19th century (in 1891 and 1892) British orchid growers placed Phalaenopsis flower stalk nodes in peat and succeeded in producing plantlets from their buds (for a review see Arditti, 1984). This method can be thought of as a crude form of tissue culture because an explant, in this case a stalk section bearing a bud cultured on a culture medium consisting of non sterile moss produced a plantlet if it did not die. This was a practical method which proved that isolated buds taken away from the plant continued to

grow. This method of orchid propagation was not noticed by botanists at the time and for many years thereafter, but at least one grower noticed it and used it. He placed sections of Phalaenopsis roots in humid enclosures and obtained a plant. This is reminiscent of micropropagation and a part of the pre-history of orchid micropropagation (Arditti and Krikorian, 1996; Arditti and Ernst, 1993; Arditti 2007). The current history of orchid micropropagation began when 1) a new [tissue culture or in vitro], simple and practical method for vegetative [clonal] propagation of Phalaenopsis [orchids] was developed at Cornell [University] five years (Rotor, 1949 cited in Arditti, 2007) before the first published report of orchid stem tip cultures (he used KC to culture Phalaenopsis nodes), and 2) Hans Thomale, a German nursery owner suggested the use of shoot tip culture for micropropagation. Dr. Rotor conceived the idea of propagating orchids while listening to Knudson lecturing on the role of sugars in plant growth. He sectioned inflorescences into nodal sections each bearing a single bud and placed on the medium. The sections grew leaves after 14-60 days. Roots were formed after 2-3 leaves were produced. Rotor wrote years later that Knudsons eyes brightened when he was shown the first successful cultures (Arditti, 1990). There can be no doubt that Dr. Gavino Rotor is the inventor of orchid micropropagation and the first to publish a paper on the subject. His method was not noticed by many at the time and few appreciated it. When it finally became known other false priority claims were widely accepted. Micropropagation of orchids: The second attempt. The history of orchid micropropagation includes controversial episodes. A history chapter in one book (Arditti and Ernst, 1993) outlined some of them incorrectly in a misguided effort not to offend. A later review (Arditti and Krikorian, 1996), is accurate. Even before the formulation of the Murashige and Skoog, medium several media were developed and used to culture a few plants including geranium (Pelargonium zonale)
* Author for correspondence: Joseph Arditti, Professor Emeritus, University of California, 4203, 5438 BS II Mail Code: 2300, Irvine, CA 92697, USA. Tel: 949 824-5221, Fax: 949 824-4709, Email: jarditti@uci.edu.

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and cyclamen (Cyclamen persicum). Hans Thomale, a German horticulturist and nursery owner and a plant scientist who later became a pharmacist, Dr. Lucie Mayer used one such medium to culture sections (Teilstcken or Pflanzenteile) and tissues (Gewebe) of orchids (for as review see Arditti and Krikorian, 1996). Thomale taught himself symbiotic and asymbiotic seed germination by reading Prof. Hans Burgeffs (1883-1976) book Samenkeimung der Orchideen. In 1946 he started a laboratory and used it to produce orchid hybrids. He carried out his orchid tissue culture research in cooperation with by Dr. Lucie Mayer. At a meeting of the Deutsche Orchideen Gesellschaft [German Orchid Society] on 23 September 1956 Thomale reported that shoot tip explants of Dactylorhiza (Orchis) maculata and some tropical orchids in vitro produced shoots and plants and stated: This is a form of vegetative multiplication whose potential cannot be overlooked. He published his findings and predictions before the first reports of Cymbidium meristem cultures by Professor Georges Morel of France who knew of Thomales work but chose not to mention it just as he did not refer to Rotors work. Thomale, did refer to Rotors work with Phalaenopsis. Thomales work did not become well known because he published his findings in German in an orchid hobby publication and in a relatively obscure book. Few scientists read his book and article. Morel aparently did but chose not to cite it, probably in an effort to aggrandize himself. Micropropagation of orchids: The third attempt. Professor Georges Morel (1916-1973) of France is widely celebrated as being the first to culture an orchid explant in vitro. This celebration is completely undeserved (Arditti and Krikorian, 1996; Arditti, 2007). In fact Morel should be criticized for not citing those whose findings he utilized in his work and for not acknowledging Rotors and Thomale's work in his initial publications. Because of that adoring hobbyists, commercial growers overwhelmed with gratitude and scientists not fully familiar with the facts elevated Morel to essentially being the sole inventor which he was far from being. Therefore it is important to note that the discovery that stem tips, root cuttings and even leaves can be used to produce healthy clones of ornamental and crop plants can be obtained from is more 50 years old (Arditti and Krikorian, 1996). Diseasefree clones of Dahlia, chrysanthemums and carnations were produced as far back as the 1940s. Given these factsm it was not surprising that Morel decided to culture shoot tips of virus infected plants in an effort to obtain disease free propagation material. Even that idea was not his. It was suggested to him by Pierre Limasset (1911-1988) and Pierre Cornuet (b. 1925). The techniques he used were also not his. They were developed in the 1940s by Professors Loo Shih Wei and Ernest A. Ball (independently of each other). Success with potatoes (and Dahlia and other plants) led Morel who was an amateur orchid grower and had in his greenhouse a Cymbidium plant of Cymbidium which was infected by Cymbidium mosaic virus to use the same tech-

nique to free it from the disease. Morel's first report on shoot tip culture of Cymbidium in the American Orchid Society Bulletin was not a reseach paper in the usual sense. It was more like a news item in a daily newspaper and referred to a non existing nutrient medium (Knudson III). The only new contribution Moral made in this paper was the term protocorm-like body, (generally abbreviated as PLB). The word protocorm was coined by Melchior Teub (1851-1910), the famed director of the Bogor Botanic Gardens in Indonesia, Melchior Treub (1851-1910; for photographs see Arditti, 1990, 1992). The first paper did not include enough information for anyone to be able to repeat the work. Only the orchid firm of Vacherot and Lecoufle La Tuilerie, Boissy-Saint Leger (Seine-et-Oise) acquired sufficient information to initiate commercial micropropagation of orchids before any other establishment. They acted quickly and had some of [their] finest cymbidiums on the market very fast. There is very little doubt (Arditti and Krikorian, 1996) that Morel gave them the information they needed and delayed publication of details to provide them with an advantage. Morel finally published his procedures, culture media and other information in some detai in the mid 1970s. However, by then the information was much less important and useful than it would have been in 1960 because others published detailed methods. Micropropagation of orchids: The fourth attempt. Dr. Donald E. Wimber (1930-1997) was an orchid cytogeneticist for the Dos Pueblos Orchid Company in Goleta, California where he studied cytology and engaged in seed germination. He learned the technique from Emil Vacin, coformulator of the Vacin and Went medium with Professor Frits W. Went, discoverer of the first plant hormone, auxin. Studies of embryonic leaves in the summer of 1955 lead him to culture them on semi-solid Vacin and Went nutrient medium. PLBs formed on the bases of these leaves. When he cut the PLBs into quarter sections and placed these on agar, the quarters produced plantlets. Wimber showed them to the owner of Dos Pueblos, Sam Mosher and the manager of the firm, Kermit Hernlund, They thought that the tissues and plantlets grew too slowly for the method to be practical and were not impressed. Wimber wrote me that he was rather fearful that some sort of chromosomal change might have occurred so that a faithful reproduction of the parent might not occur. Wimber published his first paper on clonal propagation of Cymbidium in 1963. This paper, like Morel's, was published in the American Orchid Society Bulletin, but this was the only similarity between the two papers. Wimber gave full details about his procedures, medium and culture conditions. Morel did not. Wimber credited all those whose procedures, techniques and media he used. Morel did not. Culture of Roots. The first printed suggestion to culture roots was published appeared in a theoretical article in 1970 (for a review and citation see Arditti and Krikorian, 1996).

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One of my students, Mary Ellen Farrar-Churchill cultured Epidendrum root tips in 1972, but we never obtained plantlets. Other orchid roots to be cultured were those of Phalaenopsis (in 1976, Catasetum (in 1984 for the first time), Cattleya (in 1991), Crimean orchids (2000), Cymbidium (1996), Cypripedium yatabeanum (2001), Cyrtopodium (1988), Doritaenopsis (2001), and Rhynchostylis. Rhizome tips were cultured first by Professor H. Torikata at the University of Nagoya in Japan (Ueda and Torikata, 1972; for a review see Rao, 1977). Other orchid propagated from rhizome explants followed in 1993 (Cymbidium ensifolium, Cymbidium ensifolium X Cymbidium kanran, and Cymbdium kanran Jeju X Cymbdium goeringii), 1998 (Cymbidium aloifolium, Cymbidium goeringii, Cymbdium niveo-marginatum), and 2000 (Cymbidium sinense and Geodorum densiflorum. Tuber explants were cultured for the first time by Professor Suraj Vij and Pamjab University in 1983 (Pachystoma senile). Culture of Leaves. The first protocorm-like bodies were produced by juvenile leaves in cultures derived from Cymbidium shoot tips in 1965. Protocorm production by juvenile leaves on very young seedlings lead to the development of micropropagation methods through culture of leaf bases in 1970. Epidendrum and Laeliocattleya were first propagated from leaf tips in 1971 in my laboratory. The work was carried out by Mary-Ellen Farrar-Churchill who was then an undergraduate student under joint guidance by the late Prof. Ernest A. Ball and myself. Culture of Stems. Arundina stem sections may have been first cultured around 1966. More definitive information became available in 1971. Dendrobium nodes were cultured in my laboratory in 197 Culture of flower buds, flowers, floral segments and other reproductive organs. The first orchid flower segments to be cultured were excised ovaries in 1960 by Prof. I. Ito in Japan. Prof. Ito was also the first to culture immature seeds (Dendrobium in 1955). Subsequent culture of immature seeds was of Vanilla (in 1955), Phalaenopsis (1960), Dendrobium (1961), Vanda (1964 and 1969) and Paphiopedilum (1982) The culture of immature seeds is not a method of micropropagation as such. It is sexual propagation. However since the contents of ovaries are scraped onto a culture medium it is entirely possible that some of what are presumed to be seedlings may be plantlets produced by ovary tissue and/or cells. The first young flower buds or inflorescences to be cultured were those of Ascofinetia, Neostylis and Vascostylis in 1973 in Hawaii). Inflorescences of Cymbidium, Phalaenopsis, Phragmipedium (and other orchids were cultured subsequently. Culture of Inflorescences. Rotors work pointed the way and others followed by culturing inflorescence explants of several orchids including Aranda (in 1990), Dendrobium

(1972), Doritaenopsis (1993 and 199), Mokara (1992), Oncidium (2000), and Phalaenopsis. Darkening of Culture Media. Professor John T. Curtis at the Botany Department, University of Wisconsin was the first to darken a nutrient medium. It was a medium he used for the germination of native American orchids. He used lampblack (soot produced when petroleum hydrocarbons are burned). Except for color, lampblack has nothing in common with charcoal. Charcoal used in orchid media, is made from wood, sawdust, peat and organic residues obtained during the production of pulp. It is carbonized and activated to produce a large surface area. One gram of Nuchar brand vegetable charcoal may contain up to 120 billion particles and have a total surface area of 500 to 2,000 m2. Pore distribution can range from <10 m to >500 m. The pore to volume ratio is 0.9 cc g-1. Charcoal can contain many elements, some in very small amounts. It is activated through treatment of the carbonized pyrolysis product with steam or carbon dioxide. Prof. Peter Wekmeister in Germany was the first to darken an orchid culture medium with charcoal. Robert Ernst (b.1916) was the first to add charcoal to practical seedling culture media. He found that Paphiopedilum and Phalaenopsis seedlings grew well media darkened with charcoal. These findings lead to the widespread use of charcoal-containing media for orchid seed germination, (for a review see Arditti 2007). Biotechnology. Molecular breeding (for a review see Kuehnle, 1997) of orchids started about 15 years ago through protocorm bombardment with plasmid coated microparticles: - At the University of Hawaii by A. Kuehnle and her associates - Tet Fatt Cia at the National University if Singapore and the Rockefeller University and his coworkers - Wen Huei Chen at the Taiwan Sugar corporation and his group - H. Hasebe and Tanaka in Japan H. Anzai and his collaborators in Japan\Morikawa et al used microinjection and also obtained stably transformed orchids. The use of electrophoresis by Griesbach was less successful.

REFERENCES Arditti, J. 1984. A history of orchid hybridization, seed germination and tissue culture. Bot. J. Linn. Soc. 89: 359381. Arditti, J. 1990. Lewis Knudson, his science, his times and his legacy. Lindleyana 5: 1-79. Arditti, J. 1992. Fundamentals of orchid biology. John Wiley and Sons, New York.

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Arditti, J., M.A. Clements, G. Fast, G. Hadley, G. Nishimura, and R. Ernst. 1982. Orchid seed germination and seedlingA manual. Pages 243370 in J. Arditti [ed.], Orchid Biology, Reviews and Perspectives, Vol. II. Cornell Univ. Press, Ithaca, N.Y. Arditti, J. 2007. Micropropagation of orchids. Blackwell Publishers, U. K. Arditti, J., and Ernst, R. 1993. Micropropagation of orchids. John Wiley and Sons, New York. Arditti, J., and Krikorian, A.D. 1996. Orchid micropropagation: the path from laboratory to commercialization and an account of several unappreciated investigators. Bot. J. Linn. Soc. 122: 183-241.

Chen, W.H., and Chen, H.W. 2007. Orchid biotechnology. Taiwan. Kuehnle, A.R. 1997. Molecular biology of orchids. Pages 75-115 in J. Arditti and A. M. Pridgeon (eds.), Orchid Biology, Reviews and Perspectives, Vol. VII. Kluwer Academic Publishers, Dordrecht, The Netherlands. Rasmussen, H.N. 1995. Terrestrial orchids from seed to mycotrophic plant. Cambridge University Press, Cambridge. Yam, T.W., Nair, H., Hew, C.S., and Arditti, J. 2002. Orchid seeds and their germination: An historical account. Pages 387-504 in T. Kull and J. Arditti (eds.), Orchid biology, reviews and perspectives Vol. VIII. Kluwer Academic Publishers, Dordrecht, The Netherlands.